SIST-TS CEN/TS 16835-1:2015

SLOVENSKI STANDARD
SIST-TS CEN/TS 16835-1:2015
01-september-2015
0ROHNXODUQHGLDJQRVWLþQHSUHLVNDYHLQYLWUR6SHFLILNDFLMH]DSUHGSUHLVNRYDOQH
SURFHVH]DYHQVNRSROQRNULGHO,]ROLUDQDFHOLþQD51$
Molecular in vitro diagnostic examinations - Specifications for pre-examination processes
for venous whole blood - Part 1: Isolated cellular RNA
Molekularanalytische in-vitro-diagnostische Verfahren - Spezifikationen für
präanalytische Prozesse für venöse Vollblutproben - Teil 1: Isolierte zelluläre RNS
Tests de diagnostic moléculaire in vitro - Spécifications relatives aux processus
préanalytiques pour le sang veineux total - Partie 1 : ARN cellulaire isolé
Ta slovenski standard je istoveten z: CEN/TS 16835-1:2015
ICS:
11.100.10 'LDJQRVWLþQLSUHVNXVQL In vitro diagnostic test
VLVWHPLLQYLWUR systems
11.100.30 Analiza krvi in urina Analysis of blood and urine
SIST-TS CEN/TS 16835-1:2015 en,fr,de
2003-01.Slovenski inštitut za standardizacijo. Razmnoževanje celote ali delov tega standarda ni dovoljeno.

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SIST-TS CEN/TS 16835-1:2015

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SIST-TS CEN/TS 16835-1:2015

TECHNICAL SPECIFICATION
CEN/TS 16835-1

SPÉCIFICATION TECHNIQUE

TECHNISCHE SPEZIFIKATION
July 2015
ICS 11.100.10
English Version
Molecular in vitro diagnostic examinations - Specifications for
pre-examination processes for venous whole blood - Part 1:
Isolated cellular RNA
Tests de diagnostic moléculaire in vitro - Spécifications Molekularanalytische in-vitro-diagnostische Verfahren -
relatives aux processus préanalytiques pour le sang Spezifikationen für präanalytische Prozesse für venöse
veineux total - Partie 1 : ARN cellulaire isolé Vollblutproben - Teil 1: Isolierte zelluläre RNS
This Technical Specification (CEN/TS) was approved by CEN on 30 May 2015 for provisional application.

The period of validity of this CEN/TS is limited initially to three years. After two years the members of CEN will be requested to submit their
comments, particularly on the question whether the CEN/TS can be converted into a European Standard.

CEN members are required to announce the existence of this CEN/TS in the same way as for an EN and to make the CEN/TS available
promptly at national level in an appropriate form. It is permissible to keep conflicting national standards in force (in parallel to the CEN/TS)
until the final decision about the possible conversion of the CEN/TS into an EN is reached.

CEN members are the national standards bodies of Austria, Belgium, Bulgaria, Croatia, Cyprus, Czech Republic, Denmark, Estonia,
Finland, Former Yugoslav Republic of Macedonia, France, Germany, Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania,
Luxembourg, Malta, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and United
Kingdom.





EUROPEAN COMMITTEE FOR STANDARDIZATION
COMITÉ EUROPÉEN DE NORMALISATION

EUROPÄISCHES KOMITEE FÜR NORMUNG

CEN-CENELEC Management Centre: Avenue Marnix 17, B-1000 Brussels
© 2015 CEN All rights of exploitation in any form and by any means reserved Ref. No. CEN/TS 16835-1:2015 E
worldwide for CEN national Members.

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Contents Page
Foreword .3
Introduction .4
1 Scope .5
2 Normative references .5
3 Terms and definitions .5
4 General considerations .6
5 Outside the laboratory .7
5.1 Primary venous whole blood collection manual .7
5.1.1 Information about the primary sample donor .7
5.1.2 Selection of the venous blood collection tube by the laboratory .7
5.1.3 Primary venous whole blood collection from the patient and stabilization procedures .7
5.1.4 Information on the primary blood sample and storage requirements at the blood
collection facility .8
5.2 Transport requirements .9
6 Inside the laboratory .9
6.1 Sample reception .9
6.2 Storage requirements .9
6.3 Isolation of the cellular RNA . 10
6.4 Quality assessment of isolated cellular RNA . 11
6.5 Storage of isolated cellular RNA . 11
Annex A (informative) Impact of preanalytical workflow steps on venous whole blood cellular
RNA profiles . 12
A.1 General information on operated experiments in Annex A and Annex B. 12
A.2 Influence of blood collection tube type (with or without blood cellular RNA profile
stabilizer) on the analysis of specific blood cellular RNA profiles . 12
A.2.1 Unstable blood cellular RNA profiles . 12
A.2.2 Stable blood cellular RNA profiles . 14
Annex B (informative) Influence of blood storage temperature on blood cellular RNA profiles . 16
Bibliography . 19

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Foreword
This document (CEN/TS 16835-1:2015) has been prepared by Technical Committee CEN/TC 140 “In vitro
diagnostic medical devices”, the secretariat of which is held by DIN.
Attention is drawn to the possibility that some of the elements of this document may be the subject of patent
rights. CEN [and/or CENELEC] shall not be held responsible for identifying any or all such patent rights.
According to the CEN-CENELEC Internal Regulations, the national standards organizations of the following
countries are bound to announce this Technical Specification: Austria, Belgium, Bulgaria, Croatia, Cyprus,
Czech Republic, Denmark, Estonia, Finland, Former Yugoslav Republic of Macedonia, France, Germany,
Greece, Hungary, Iceland, Ireland, Italy, Latvia, Lithuania, Luxembourg, Malta, Netherlands, Norway, Poland,
Portugal, Romania, Slovakia, Slovenia, Spain, Sweden, Switzerland, Turkey and the United Kingdom.
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Introduction
Molecular in vitro diagnostics has enabled a significant progress in medicine. Further progress is expected by
new technologies analyzing signatures of nucleic acids, proteins, and metabolites in human tissues and body
fluids. However, the profiles of these molecules can change drastically during primary sample collection,
transport, storage, and processing thus making the outcome from diagnostics or research unreliable or even
impossible because the subsequent analytical assay will not determine the situation in the patient but an
artificial profile generated during the pre-examination process. Therefore, a standardization of the entire
process from sample collection to RNA analysis is needed. Studies have been undertaken to determine the
important influencing factors. This Technical Specification draws upon such work to codify and standardize the
steps for venous whole blood cellular RNA analysis in what is referred to as the preanalytical phase.
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1 Scope
This Technical Specification recommends the handling, documentation and processing of venous whole blood
specimens intended for cellular RNA analysis during the preanalytical phase before a molecular assay is
performed. This Technical Specification covers specimens collected by venous whole blood collection tubes.
This Technical Specification is applicable to molecular in vitro diagnostic examinations (e.g. in vitro diagnostic
laboratories, laboratory customers, in vitro diagnostics developers and manufacturers, institutions and
commercial organizations performing biomedical research, biobanks, and regulatory authorities).
Blood cellular RNA profiles can change significantly after collection. Therefore, special measures need to be
taken to secure good quality blood samples for cellular RNA analysis and storage.
Different dedicated measures need to be taken for stabilizing blood cell free circulating RNA and RNA in
exosomes circulating in blood, which are not described in this Technical Specification.
Different dedicated measures need to be taken for collecting, stabilizing, transporting and storing capillary
blood as well as for collecting and storing blood by paper based technologies. These are not described in this
Technical Specification.
RNA in pathogens present in blood is not covered by this Technical Specification.
2 Normative references
The following documents, in whole or in part, are normatively referenced in this document and are
indispensable for its application. For dated references, only the edition cited applies. For undated references,
the latest edition of the referenced document (including any amendments) applies.
EN ISO 15189:2012, Medical laboratories - Requirements for quality and competence (ISO 15189:2012,
Corrected version 2014-08-15)
ISO 15190, Medical laboratories — Requirements for safety
3 Terms and definitions
For the purposes of this document, the terms and definitions given in EN ISO 15189:2012 and the following
apply.
3.1
ambient temperature
unregulated temperature of the surrounding air
3.2
analytical phase
processes that start with the isolated analyte and include all kind of parameter testing or chemical
manipulation for quantitative or qualitative analysis
3.3
blood cellular RNA
cellular RNA
RNA molecules present in blood cells
3.4
blood cellular RNA profile\s
amounts of different RNA molecules, that are present in blood cells and that can be measured in the absence
of any losses, inhibition and interference
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3.5
blood cellular RNA profile stabilizers
compounds, solutions or mixtures that are designed to minimize changes of the blood cellular RNA profile
3.6
pre-examination processes
preanalytical phase
preanalytical workflow
processes that start, in chronological order, from the clinician’s request and include the examination request,
preparation and identification of the patient, collection of the primary sample(s), temporary storage,
transportation to and within the analytical laboratory, aliquotting, retrieval, isolation of analytes, and end when
the analytical examination begins
[SOURCE: EN ISO 15189:2012, 3.15, modified — An additional term was added and more details were
included.]
Note 1 to entry: The preanalytical phase may include preparative processes that may influence the outcome of the
intended examination.
3.7
primary sample
specimen
discrete portion of a body fluid, breath, hair or tissue taken for examination, study or analysis of one or more
quantities or properties assumed to apply for the whole
[SOURCE: EN ISO 15189:2012, 3.16, modified — The term and definition is used here without the original
notes.]
3.8
RNA
ribonucleic acid
polymer of ribonucleotides occurring in a double-stranded or single-stranded form
[SOURCE: EN ISO 22174:2005, 3.1.3]
3.9
room temperature
temperature which is defined as 18 °C to 25 °C for the purposes of this document
3.10
stability
ability of a sample material, when stored under specified conditions, to maintain a stated property value within
specified limits for a specified period of time
[SOURCE ISO Guide 30:1992, 2.7]
Note 1 to entry: The measured constituent for the purpose of this document is blood cellular RNA.
4 General considerations
For general statements on primary sample collection and handling (including avoidance of cross
contaminations), see EN ISO 15189:2012, 5.2.6, 5.4.4. Consumables including kits shall be verified before
use in examination (see EN ISO 15189:2012, 5.3.2.3); EN ISO 15189:2012, 5.5.1.2 and 5.5.1.3 can also
apply.
As all steps of a diagnostic workflow can influence the final analytical performance, the entire workflow
comprising the preanalytical steps, including information on sample stability and storage conditions, and the
analytical steps should be verified and validated (see EN ISO 15189).
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Blood cellular RNA profiles can change significantly after collection (e.g. gene induction, gene down
regulation, RNA degradation) [3], [4], [5], [6]. These changes can vary individually in different blood donors’ /
patients’ blood [3], [7], [8], [9], [10].
The stability of the specific blood cellular RNA profile of interest should be investigated throughout the
complete preanalytical workflow.
Before or during the design of the analytical test system it should be investigated and ensured that the specific
blood cellular RNA molecule/s amount/s intended to be analyzed in the analytical test is/are not affected by
the envisioned entire preanalytical workflow.
If a commercial product is not used in accordance with the manufacturer's instructions, responsibility for its
validation, verification, use and performance lies with the user.
Safety regulations on transport and handling shall be considered (EN ISO 15189:2012, 5.4.5 and ISO 15190).
5 Outside the laboratory
5.1 Primary venous whole blood collection manual
5.1.1 Information about the primary sample donor
The documentation should include, but is not limited to:
a) the primary donor / patient ID, which can be in the form of a code;
b) the health status and relevant lifestyle factors of the blood donor (e.g. healthy, disease type, diet, gender,
age);
c) the information about medical treatment and special treatment prior to blood collection (e.g. anaesthetics,
medications, fasting status);
d) the type and purpose of the analytical test requested.
See also EN ISO 15189:2012, 5.4.4.
5.1.2 Selection of the venous blood collection tube by the laboratory
Due to the high instability of blood cellular RNA profiles in individual patients/donors [3], [7], [8], [9], [10],
commercially available blood collection tubes containing blood cellular RNA profile stabilizers should be used
[7], [8], [10], [11], [12] (Figure A.1).
Blood collection tubes not containing any blood cellular RNA profile stabilizer should only be used, if the
specific blood cellular RNA molecule or the blood cellular RNA profile to be analyzed is stable after blood draw
(Figure A.2) or if the requested analytical test allows the use of such tubes.
5.1.3 Primary venous whole blood collection from the patient and stabilization procedures
1. The identity of the person collecting the sample and the time of blood collection according to
EN ISO 15189:2012, 5.4.4.3, f) shall be documented.
2. For the labelling (sample identification) of the blood collection tube a routine procedure
(EN ISO 15189:2012, 5.4.4.3, e)) or a procedure with additional information (e.g. 2D-barcode) shall be
used.
3. Standard venepuncture technique can be used. Steps for preventing possible backflow may be
required. The manufacturers’ instructions for using the blood collection tubes shall be followed. A
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blood collection set and needle holder can be required when using blood cellular RNA profile stabilizer
containing tubes. In this case, the instructions of the collection set and needle holder manufacturer
shall be followed.
NOTE There is no known specific effect of venous whole blood draw procedure on the cellular RNA. Routine
procedures can therefore be used.
4. Blood collection tubes shall be filled in accordance to the manufacturers’ instructions and attention
should be drawn to the correct positioning of the collection tube during the blood draw as well as the
required volume.
5. Blood collection tube manufacturers' instructions, for mixing or inverting the tube immediately after
blood collection, shall be followed.
NOTE Unless additives are homogenously mixed with the blood sample, the blood cellular RNA profile
quality and the quality of individual cellular RNA molecules can be compromised, which can impact the validity
and reliability of the analytical test results.
6. Any tampering with and/or additions to the primary sample shall be documented.
5.1.4 Information on the primary blood sample and storage requirements at the blood collection
facility
The documentation on the primary blood sample shall include the date and time of blood collection [3], [14],
[15], [16] as blood cellular RNA profiles can change significantly after blood collection and can thereby affect
the validity and reliability of the analytical test result [3], [11], [13],[14].
For storing the primary blood samples collected in blood collection tubes with blood cellular RNA profile
stabilizers, the blood collection tube manufacturers' instructions on storage conditions shall be followed
(temperature and storage duration). Where the analytical test providers' instructions are more stringent, these
shall be followed. The storage conditions (storage duration and temperature) shall be documented.
Blood collection tubes without blood cellular RNA profile stabilizers should only be used, if the ordered
analytical test specifications allow the usage of such tubes. In these cases, the analytical test providers'
instructions on storage conditions shall be followed. This can require documentation of storage conditions.
When using blood collection tubes without blood cellular RNA profile stabilizers and no requirements on the
storage conditions are available, the primary blood samples should be transferred immediately to 2 °C to 8 °C
or on wet-ice in order to minimize blood cellular RNA profile changes [8], [14], [15], [16] (Figure B.1). The
storage conditions (storage duration and temperature) shall be documented. The storage duration allowed at
2 °C to 8 °C or on wet-ice is highly dependent on the stability of the individual RNA molecules and their
cellular quantities to be analyzed in the analytical test. This stability can vary between several minutes and
over 24 h.
NOTE Under these storage conditions (at 2 °C to 8 °C or on wet-ice) blood cellular RNA profile changes can still
occur when no blood cellular RNA profile stabilizers are used (Figure B.1).
For samples dedicated to be archived in a biobank it is usually not known which individual RNA molecules will
be analyzed after archiving, therefore tubes without blood cellular RNA profile stabilizers should not be used
for biobanking.
The temporary storage duration in the blood collection facility contributes to the total duration for storage.
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5.2 Transport requirements
The required transport conditions shall be documented including any deviations.
When using blood collection tubes with blood cellular RNA profile stabilizers, the tubes' manufacturers'
instructions on transport conditions shall be followed (e.g. temperature, transport duration). Where the
analytical test providers' instructions are more stringent, these shall be followed.
When using blood collection tubes without blood cellular RNA profile stabilizers, the analytical test providers´
instructions on transport conditions shall be followed. This can require the documentation of transport
conditions (duration and temperature).
When using blood collection tubes without blood cellular RNA profile stabilizers and no analytical test
provider's instructions are available, the primary blood sample should be transported at 2 °C to 8 °C or on wet-
ice without delay in order to minimize the blood cellular RNA profile changes [16].
See also EN ISO 15189:2012, 5.4.5.
The transport duration to the laboratory contributes to the total duration for storage.
6 Inside the laboratory
6.1 Sample reception
The blood sample reception time shall be documented. Nonconformities of labelling, transport conditions and
blood volume differences to specifications, leaking/broken tubes, etc. shall be documented.
Where there are nonconformities in labelling, transport conditions, overall storage and transport duration or
blood volume that could affect the validity and reliability of the analytical test result, a new sample should be
obtained.
6.2 Storage requirements
The storage temperature and time interval between sample receipt and sample processing for cellular RNA
isolation shall be documented. Storage temperature and total storage duration shall not exceed specifications
identified in 5.1.4 and 5.2.
The primary blood sample total storage duration shall include the duration for storage at the blood collection
facility (5.1.4), for transportation to the laboratory (5.2) and for further storage at the laboratory or other
institutions.
The stability of specific blood cellular RNA profiles can vary between different RNA molecules (e.g. mRNA,
rRNA, miRNA) different blood donors, and different blood storage conditions. Any specified maximum storage
duration given by the blood collection tube manufacturer or the analytical test manufacturer shall not be
exceeded. If such specifications are not available, the maximum storage duration shall be validated and
generally kept to a minimum.
See also Table 1.
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Table 1 — Summary of storage conditions for venous whole blood collection tubes with or without
blood cellular RNA stabilizers
Blood collection tube Blood collection, transport and storage
Duration Temperature
With blood cellular RNA profile — Blood collection tube manufacturer’s instructions
stabilizer
a
— Analytical test providers' instructions
Without blood cellular RNA — Analytical test instructions — Analytical test instructions
profile stabilizer
b
— Wet-ice or 2 °C to 8°C
a
If more stringent than blood tube manufacturer’s instructions.
b
Under these conditions blood cellular RNA profile changes can still occur (Figure B.1).
6.3 Isolation of the cellular RNA
The laboratory shall validate the entire process comprising sample collection, storage, transportation and
isolation of cellular RNA to the final analytical test result according to its internal quality management system
(see EN ISO 15189).
To avoid a cross contamination with amplified RNA, the isolation of the cellular RNA should not be performed
in the same area as the amplification, unless a closed system is used.
When processing blood from tubes containing blood cellular RNA profile stabilizers, kits specified by the
manufacturer of the blood collection tube should be used for the isolation of cellular RNA. Alternative
extraction procedures can be used if verified for the same requirements and validated for the same intended
use.
NOTE 1 When using alternative extraction procedures, dedicated measures and technologies might be needed in
order to avoid carrying over cellular RNA stabilization molecules to the final RNA eluate. Carry-over from stabilizer
molecules can lead to an inhibition of the analytical test reaction.
Blood collection tube and kit manufacturers’ instructions or the instructions for the validated alternative for
isolating the cellular RNA shall be followed.
NOTE 2 Dedicated procedures for use for processing frozen blood samples can be included in the manufacturers’
instructions.
When using blood collection tubes not containing any blood cellular RNA profile stabilizer, the analytical test
manufacturers’ instructions or validated alternatives for cellular RNA isolation shall be followed.
When using blood collection tubes not containing any blood cellular RNA profile stabilizer, where there are no
analytical test manufacturers' instructions, the laboratory shall validate the entire cellular RNA isolation
process.
A DNase treatment step should be incorporated into the cellular RNA isolation procedure [17], [18]. The
DNase, other reagents and consumables coming in contact with the cellular RNA sample should be RNase-
free.
If blood collection tubes containing frozen samples without any blood cellular RNA profile stabilizer are used,
thawing should be avoided as this can lead to immediate degradation of cellular RNA. To limit cellular RNA
degradation, the frozen blood should be transferred from the blood collection tube directly into a blood cellular
RNA profile stabilizing extraction buffer and immediately be homogenized. The homogenized sample should
be further cleaned up and concentrated by validated protocol steps. The extracted RNA should be kept at
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2 °C to 8 °C (e.g. cooling block) or on wet-ice and should be assayed immediately. For longer storage and
archiving see 6.5.
6.4 Quality assessment of isolated cellular RNA
The RNA quantity and quality should be checked according to diagnostic kit manufacturer’s instructions, or
according to validated procedures by generally accepted physical, chemical and biochemical procedures [13],
[19], [20]. These may include one or more of the following:
a) quantification by absorption (A ) or spectrofluorometry;
260
b) test for purity by absorption measurements (wavelength scan, A /A ratio);
260 280
c) test for RNA integrity (by electrophoresis, chromatography, or molecular methods such as the 3´/5´assay
or differential length amplicon ratio[21]);
d) test for presence of interfering substances (using exogenous controls (spiked in RNA and DNA controls)
or inspecting qPCR response curves for anomalies [22]).
The cellular RNA isolation performance should be tested in a RNA proficiency test program.
6.5 Storage of isolated cellular RNA
For storing and archiving the isolated cellular RNA (before the analytical phase), the RNA isolation kit
providers’ specific instructions should be followed.
If there is no information available from the RNA isolation kit provider or if laboratories’ own validated cellular
RNA isolation procedures are used, the isolated cellular RNA should be assayed immediately. In case that the
isolated cellular RNA cannot be assayed immediately, the laboratory shall have verified protocols in place how
to store the isolated cellular RNA.
Storage on wet-ice for a short period of time (i.e. 30 min) can be appropriate in certain circumstances.
Storage for archiving purposes should be at −70 °C or below [16].
Some cellular RNA isolation procedures may allow archiving the isolated cellular RNA at −20 °C to −70 °C.
For archiving, aliquots of the isolated cellular RNA should be generated to avoid freezing and thawing.
Unintended freeze-drying of the isolated cellular RNA during archiving due to water evaporation should be
avoided as the RNA can degrade and the recovery from the tube can be difficult or even impossible.
Therefore, appropriate tubes avoiding water evaporation during archiving should be used.
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Annex A
(informative)

Impact of preanalytical workflow steps on venous whole blood cellular
RNA profiles
A.1 General information on operated experiments in Annex A and Annex B
1)
SPIDIA (Standardisation and Improvement of
...